Hereditary spherocytosis. II. Observations on the role of the spleen

1. Three patients with hereditary spherocytosis and 1 patient with the Fanconi syndrome (pancytopenia and multiple congenital abnormalities) were transfused prior to splenectomy with normal erythrocytes of types which could bedifferentiated serologically from those of the recipients. The proportionsof donated and patient’s cells in peripheral blood and in blood washed from theminced spleens were determined by differential agglutination, and the osmoticand mechanical fragilities of the two types of cells in peripheral and splenic bloodwere measured by differential agglutination of the corpuscles remaining in eachtest tube after partial hemolysis had occurred.

2. In each case of hereditary spherocytosis the proportion of recipient’s cellswas much higher in splenic than in peripheral blood, indicating selective retentionof the thicker corpuscles within the spleen. Osmotic fragility of thie patient’s redcells was much greater in samples of splenic mince blood than in peripheral venoussamples, while the fragility of the donated red corpuscles was normal or nearlynormal in both splenic and peripheral blood.

In the patient exhibiting the Fanconi syndrome, on the other hand, neitherthe patient’s red cells nor donated red cells were retained to any extent in thespleen and the fragility of neither type of cell was altered.

3. Spleens removed surgically from 3 patients with idiopathic thrombocytopenic purpura were perfused with mixtures of normal A or B cells and groupO cells drawn from a splenectomized individual with hereditary spherocytosis.During perfusion the spheroidal cells were selectively removed from the mixtures and at the end of each experiment red cells of this type predominated inthe blood samples washed from the minced spleens. A fourth excised spleen wasperfused with a mixture of two types of normal cells, neither of which was retained to any extent by the spleen during perfusion. The perfusion experimentsshow that spleens from patients with nonhemolytic disease are also capable ofselective trapping of spheroidal cells.

4. The experiments described indicate that the spleen acts as a filter and trapand as an "incubator" in accelerating destruction of red corpuscles in patientswith hereditary spherocytosis.

Submitted on April 16, 1951 Accepted on May 10, 1951     

Effect of stasis of blood in varicose veins on erythrocyte fragility,with accompanying studies comparing red cells and other blood elements with cubital vein blood

1. Blood from varicose veins was compared with cubital vein blood in 20 patientsin order to determine whether or not the degree of stasis present in varicose veinswould increase red cell fragility. Corollary studies consisted of comparative determinations of red cells, hemoglobin, packed cell volume, white blood cells,platelets and serum proteins.

2. There was no increase in red cell fragility in the varicose vein specimen, indicating that the theory that minor degrees of intravascular erythrostasis contribute substantially to some of the hemolytic anemias is untenable.

3. There was a small but statistically significant elevation in red cells per cu.mm. in varicose vein blood as compared with blood from cubital veins. There wasa suggestive, but not significant, increase in packed cell volume and serum proteinin the varicose vein samples. The evidence indicates a mild degree of hemoconcentration.

4. White cells, platelets and hemoglobin determinations were found to have thesame values in varicose vein blood as in blood from the cubital vein.

     

Studies on the Formation of Heinz Bodies. II. The Nature and Significance of Heinz Bodies

Simultaneous studies were made as to the degrees of Heinz body formation,of change in hemoglobin and of change in osmotic fragility in erythrocytes incubated with certain drugs which are known to produce Heinz bodies in vivo.Certain factors that modify the pattern of effect of drugs on normal adult erythrocytes were also studied.

A close relationship was found between the degrees of Heinz body production and "intact" hemoglobin destruction. Factors that caused variation in thedegree of "intact" hemoglobin destruction caused similar variation in thedegree of Heinz body production. Staining studies suggested that Heinzbodies are protein in nature and derived from hemoglobin. These observationswere taken to indicate that Heinz bodies probably result from the direct effectof certain drugs on intra-erythrocytic hemoglobin.

-naphthol and primaquine were found to cause marked increase in osmoticfragility relative to the degrees of Heinz body production and of "intact" hemoglobin destruction, whereas acetyl phenylhydrazine produced marked Heinzbody production and "intact" hemoglobin destruction with relatively littleincrease in osmotic fragility. These observations were regarded as evidencethat Heinz body production is not the sole determinant of instability of erythrocytes exposed to these various drugs.

Under no experimental conditions was Heinz body production or "intact"hemoglobin destruction found without methemoglobin formation. Evidencewas presented to indicate that Heinz body production could be inhibited bytrapping methemoglobin as methemoglobin cyanide or methemoglobin azide.The suggestion was advanced that methemoglobin is in fact an essential stagein the destruction of "intact" hemoglobin and the formation of Heinz bodies.

Submitted on October 6, 1960 Accepted on January 12, 1961     

Hereditary nonspherocytic hemolytic disease; a study of a singular familial hemolytic syndrome

Extensive studies were performed on four cases from three unrelated kindredswith a familial hemolytic syndrome not associated with any significant red cellanomaly (hereditary nonspherocytic hemolytic disease). These cases were compared with similar ones already reported in the literature.

1. Hereditary nonspherocytic hemolytic disease appears to be transmitted asa Mendelian dominant. Frequently the gene responsible for the condition seemsto have low expressivity. In some cases, the hereditary mechanism may be dueto inheritance of a recessive gene from each parent. The basic erythrocytic defectresponsible for the condition is unknown. In view of various clinical and hematologic findings, it is likely that hereditary nonspherocytic hemolytic disease maybe a group of diseases involving more than one mechanism.

2. All criteria of hemolytic anemia (erythroid hyperplasia of the bone marrow,reticulocytosis, hyperbilirubinemia, increased fecal urobilinogen, rapid turnoverof tracer iron in the plasma) were satisfied.

3. Red cell survival time studies revealed an intraerythrocytic defect with amean life span of twelve to seventeen days. Normal red cells transfused into thepatients under study survived normally. Anemia was normochromic and normocytic or macrocytic; it varied from mild to severe.

4. Osmotic and mechanical fragility of the red cells was normal. Osmotic andmechanical fragility tests after incubation at 37 C. for 24 hours in some showed amild increase compared with normal controls. Autohemolysis of incubated oxalated blood was not marked and varied from case to case.

5. The electrophoretic mobility of hemoglobin from the patients was that ofnormal adult hemoglobin. Small increases of fetal hemoglobin were seen in several cases.

6. In contrast to the histologic findings in hereditary spherocytosis the splenicpulp was not congested, but hemosiderin deposits were heavy. Liver biopsy specimens showed deposits of hemosiderin in parenchymal and Kupffer cells.

7. Splenectomy did not arrest the hemolytic process. Mild improvement was seenin one case. In most cases the operation is of no value.

8. Diagnostic difficulties may be encountered with mild cases of hereditaryspherocytosis. Examination of rouleaux in fresh blood and an osmotic fragilitytest in 0.65 per cent sodium chloride after incubation usually establishes the differential diagnosis. The condition may present clinically as hemolytic disease ofthe newborn and must be differentiated from erythroblastosis due to Rh or otherblood group incompatibilities. Other hereditary hemolytic diseases such assickle cell anemia, Cooley’s anemia, hereditary spherocytosis, and hereditaryhemolytic elliptocytosis are easily ruled out by their typical clinical and hematologic manifestations. When a family study is negative or cannot be done, a redcell survival time determination may be necessary to rule out acquired hemolyticanemia with a negative Coombs test. Some cases that have been diagnosed asconstitutional hyperbilirubinemia (familial nonhemolytic jaundice) may actuallyrepresent mild hereditary nonspherocytic hemolytic disease.

Submitted on October 7, 1953 Accepted on November 3, 1953     

Hereditary Stomatocytosis With Hemolytic Anemia in the Dog

An autosomal recessive mutant (symbol,dan) in the dog causes chondrodysplasticdwarfism and mild anemia as pleiotropiceffects. The anemia is characterized bystomatocytosis, macrocytosis, low MCHC,increased osmotic fragility, shortened redcell survival, reticulocytosis, erythroidhyperplasia, and increased iron turnover.Minor changes can be detected in the redcells of heterozygous carriers of the dangene, but red cell survival is normal andchondrodysplasia is not present. Red cellsodium concentration and water contentare increased in anemic dogs and to alesser degree in carriers. Glutathione deficiency is present. The disorder describedhere resembles a number of human hemolytic anemias with stomatocytosis, glutathione deficiency, and disordered red cellcation and water content, and furtherstudy of the dan mutant, together withinvestigations of the hereditary humanconditions, will elucidate the maintenanceof normal red cell cation balance.

Submitted on January 15, 1974 Accepted on April 18, 1974     

Adverse Effect of Raised Environmental Temperature on the Expression of Hereditary Spherocytosis in Deer Mice

Deer mice with a compensated hemolytic syndrome caused by hereditaryspherocytosis were placed in a controlled temperature room at 35 C. Severeanemia, red cell fragmentation, increased osmotic fragility, occasional jaundiceand hemoglobinuria were observed. The spleen enlarged and the erythropoietic bone marrow expanded. Reticulocytes fell markedly early during heat exposure, but in survivors soon increased to levels higher than those observedprior to experiment. Mortality from the severe acceleration of the preexistinghemolysis was high. Control nonspherocytic deer mice subjected to an identical temperature showed only a mild drop of hemoglobin and slight elevationof reticulocytes.

Since hereditary spherocytosis in deer mice in a temperate climate is compatible with good health, these findings provide a further illustration of interaction of a particular environmental insult with a specific genotype in thepathogenesis of hereditary disease.

Submitted on December 10, 1965 Accepted on January 10, 1966     

Hereditary spherocytosis. I. Clinical,hematologic and genetic features in 28 cases,with particular reference to the osmotic and mechanical fragility of incubated erythrocytes

Clinical, hematologic and genetic data on 28 cases of hereditary spherocytosisare presented for the purpose of characterizing this disorder as completely aspossible. On the basis of this experience it is recommended that the followingtypical laboratory findings be sought in establishing a diagnosis in suspectedcases: (1) Presence of spherocytes or abnormally thick red cells in peripheralblood; (2) greater than normal osmotic fragility of the red cells; in cases in whichthe fragility of fresh cells is not significantly increased, determinations should bemade after sterile incubation of the blood at body temperature for 24 hours;(3) greater than normal mechanical fragility of freshly drawn red cells; (4) negative antiglobulin (Coombs) test; (5) greater than normal lysis of the red cellsduring sterile incubation at body temperature for 48 hours; and (6) presence ofsimilar abnormalities in relatives.

Abnormality of the erythrocyte persisted in all of the 11 patients in this seriesfollowed one or more years after splenectomy. An unusual case of chronic hemolytic anemia is described but not included in the numbered series because (1) bothparents were hematologically normal and (2) spherocytosis and abnormally greatosmotic and mechanical fragility and autohemolysis could not be demonstratedafter the fifth postoperative month. Classification of this case is deferred pendingfurther experience.

Demonstration in a parent, sibling or offspring of red cells showing the afore-mentioned abnormalities is necessary for an unequivocal diagnosis, but this requirement cannot always be met because relatives may not be available for examination. Moreover, when parents and/or several siblings are examined withoutpositive findings, low gene expressivity, gene mutation and illegitimacy may beconsidered as explanations. Evidence is cited to suggest the possibility of a lowdegree of penetrance or expression in some cases and to illustrate the need forstill more sensitive laboratory tests that might aid in diagnosis of the mildestforms of this disease. The lower incidence of spherocytosis in siblings of propositithan in offspring of propositi is cited as evidence bearing on the theory of genemutation in some propositi.

A simplified "qualitative" test of osmotic fragility of incubated red cells isdescribed.

Submitted on April 16, 1951 Accepted on May 10, 1951     

Erythrocyte osmotic fragility and oxidative stress in experimental hypothyroidism

The present study was planned to explain the relation between erythrocyte osmotic fragility and oxidative stress and antioxidant statue in primary hypothyroid-induced experimental rats. Twenty-four Spraque Dawley type female rats were divided into two, as control (n=12) and experimental (n=12), groups weighing between 160 and 200 g. The experimental group animals have received tap water methimazole added standard fodder to block the iodine pumps for 30 d (75 mg/100 g). Control group animals were fed tap water and only standard fodder for the same period. At the end of 30 d blood samples were drawn from the abdominal aorta of the rats under ether anesthesia. T₃, T₄, and TSH levels were measured and the animals that had relatively lower T₃, T₄, and higher TSH levels were accepted as hypothyroid group. Hormone levels of the control group were at euthyroid conditions. Osmotic fragility, as a lipid peroxidation indicator malondialdehyde (MDA), antioxidant defense system indicators superoxide dismutase (SOD) and glutathione (GSH) levels were measured in the blood samples. Osmotic fragility test results: There was no statistically significant difference found between maximum osmotic hemolysis limit values of both group. Minimum osmotic hemolysis limit value of hypothyroid group was found to be higher than that of control group values (p<0.02). The standard hemolysis and hemolytic increment curve of the hypothyroid group drawn according to osmotic fragility test results was found to be shifted to the right when compared to control group’s curve. This situation and hemolytic increment value, which shows maximum hemolysis ratio, is the proof of increased osmotic fragility of the erythrocytes in hypothyroidism. There is no statistically significant difference found between hypothyroid and control groups in the lipid peroxidation indicator MDA and antioxidant indicators SOD and GSH levels. As a result of our study it may be concluded that hypothyroidism may lead to an increase in osmotic fragility of erythrocytes. But the increase in erythrocyte osmotic fragility does not originate from lipid peroxidation.     

Flow Cytometric Eosin-5′-Maleimide Test is a Sensitive Screen for Hereditary Spherocytosis

Hereditary spherocytosis (HS) is a clinically heterogeneous disease characterized by mild to moderate hemolysis resulting from red cell membrane protein defects. Diagnostic tests include hemogram, reticulocyte count and blood smear evaluation, osmotic fragility, cryohemolysis, SDS-PAGE, flow cytometry using eosin-5′-maleimide (EMA) and genetic studies. We evaluated the flow cytometric EMA-binding test and compared it with osmotic fragility in 51 consecutive cases of suspected HS aged between 10 days and 62 years. In addition, 4 cases suspected on blood smears underwent EMA testing alone. The 16 EMA-positive cases who were determined to have HS had overlapping hemoglobin levels and reticulocyte counts with the 35 patients with normal EMA results, highlighting the importance of the flow cytometric test in providing a definitive diagnosis. Flow cytometric EMA binding test was thus a simple and relatively faster method to confirm HS in our experience.     

Hemoglobin Function in the Horse: The Role of 2,3-Diphosphoglycerate in Modifying the Oxygen Affinity of Maternal and Fetal Blood

The blood of the newborn horse was foundto have a higher affinity for oxygen thanthat of the mother. This difference was dueto the fact that the red cells of newbornfoals contained 36% lower 2,3-diphosphoglycerate (2,3-DPG) than red cells fromtheir respective mares. The ATP levels offoal and maternal red cells did not differsignificantly. Following birth a prompt risein the foal's red cell 2,3-DPG occurred,approaching normal (maternal) levelswithin 5 days. Unlike many other species,the hemoglobins of the newborn and adulthorse have been shown to be structurallyidentical. Furthermore, phosphate-freesolutions of newborn and maternal hemoglobins had identical oxygen saturationcurves in the absence and presence ofadded 2,3-DPG. This study demonstratesthat, in contrast to other species. the increased oxygen affinity of horse fetal redcells is due to a lower level of the cofactor2,3-DPG rather than to the presence offetal hemoglobin.

Submitted on November 28, 1972 Revised on February 17, 1973 Accepted on March 5, 1973     

Erythrocyte osmotic fragility of rats receiving the thermostable plasma erythropoietic factor

1. The thermostable erythropoietic factor in "anemic" rabbit plasma stimulates cellular division of marrow erythrocytic precursors in the normal rat producing erythrocytosis due to microcytes without evidence of augmented hemoglobin synthesis.

2. Study of erythrocyte osmotic behavior in these animals with a quantitative photocolorimetric method failed to demonstrate significant variation fromthe controls.

3. Osmotic fragility determinations by a direct cell enumeration technic revealed decreased resistance of the microcytes to lysis in hypotonic sodiumchloride solutions.

4. It is concluded from these data that the erythrocytes in the normal ratinjected with the heat stable plasma erythropoietic factor comprise two distinctcell types. They are microcytes with shortened survival times and normalerythrocytes.

5. The decreased viability of these microcytes is the apparent explanationfor the rapid re-establishment of normal erythrocytic equilibrium after the"anemic" plasma extract injections are stopped.

Submitted on July 1, 1957 Accepted on September 15, 1957     

Red cell osmotic fragility studies in hemoglobin C‐β thalassemia: osmotically resistant microspherocytes

Typically certain features of red cell morphology predict the results of osmotic fragility testing. Microspherocytes generally have increased and target cells decreased fragility. Blood smears in homozygous hemoglobin C disease show an interesting admixture of microspherocytes and target cells. Yet osmotic fragility studies generally show only reduced fragility and no population of fragile cells to correspond with the spherocytes. The present study demonstrates that the red cells of patients with hemoglobin C‐β thalassemia share many characteristics with hemoglobin C red cells, including the decreased osmotic fragility of all cells despite the presence of both spherocytes and target cells. These paradoxically osmotically resistant spherocytes probably arise because of cellular dehydration due to a K‐Cl transport system which may be activated by binding of hemoglobin C to the red cell membrane.     

Sickle Cell--Hemoglobin C Disease: Quantitative Determination of Iron Kinetics and Hemoglobin Synthesis

Quantitative determinations of iron kinetics and hemoglobin synthesis weremade on five patients with sickle cell-hemoglobin C disease. The anemia wasmild in all patients but one who had the hemoglobin of 10.1 Gm. per cent.All patients were in a steady state during the period of this study.

The ferrokinetic determinations demonstrated a hemolytic process in allcases. The mean erythrocyte life span in these patients was 18, 20, 29, 46and 56 days respectively (normal range, 110-130 days). The hemoglobinsynthesis was increased in all. Reduced to terms of daily hemoglobin production per liter of blood, the values were 2.2 Gm., 2.9 Gm., 3.6 Gm., 5.8 Gm.and 6.6 Gm. The latter figure represents a five fold increase over the normalmean value of daily hemoglobin synthesis of 1.3 Gm. (normal range, 1.0 to1.6 Gm. of hemoglobin per liter of blood per day).

The results of the in vivo organ counts demonstrated a significant degreeof sequestration of red blood cells in the spleen of two patients. The questionof advisability of splenectomy in such patients was discussed.

Accepted on December 3, 1962     

Studies on erthrocytes in cases with past history of favism and drug-induced acute hemolytic anemia

Studies have been carried out on erythrocytes of cases with past history ofacute hemolysis due to Favism, sulpha drugs or PAS.

No indication was found of abnormal osmotic or mechanical fragility, electrophoretic mobility of hemoglobin or its amino acid content. Similarly no abnormal glycolytic or catalase activities were detected.

Glutathione (GSH) deficiency of erythrocytes was discovered in all casesstudied. Similarly, glutathione (GSH) deficiency was a frequent finding amongthe relatives of affected cases.

No such glutathione deficiency could be established in the cases of thalassemiamajor so far studied, though both patient-groups without any exception belongto the same ethnographic denomination (non-Ashkenazic).

This investigation was aided by a grant from the Israel Research Council.

Submitted on October 10, 1956 Accepted on November 26, 1956     

Studies on spontaneous in vitro autohemolysis in hemolytic disorders

Measurements of spontaneous lysis (autohemolysis) of red cells in sterile defibrinated blood after 48 or more hours of incubation at body temperature werefound useful in the investigation of certain hemolytic states. Abnormally rapidautohemolysis was demonstrated most consistently in hereditary spherocytosis,but was also found in other types of spherocytosis and in several examples ofnon-spherocytic hemolytic anemia. Autohemolysis above the normal range canbe regarded as strong presumptive evidence of a hemolytic disorder, but a normalrate of autohemolysis by no means excludes the possibility of a hemolytic process in any given case.

Abnormalities of hereditary spherocytes causing increased autohemolysis wereshown to be correlated with those responsible for spherocytosis as reflected inosmotic fragility tests. There appeared to be closer correlation with abnormalities causing increased osmotic fragility of the cells after 24 hours incubation.The autohemolysis test and the osmotic fragility test on incubated red cells werefound to be equally sensitive in their capacity to detect spherocytosis of thehereditary type.

Addition of adenosine, guanosine or inosine caused moderate to marked reduction in autohemolysis of nearly all types of red cells tested. Lysis of incubatedspherocytes from active cases of autoimmune hemolytic disease was more markedly inhibited by a small amount of adenosine than was the lysis of hereditaryspherocytes.

Glucose regularly caused marked inhibition of autohemolysis of hereditaryspherocytes and of red cells from patients with acute leukemia. The effect ofglucose on HS red cells might have been due in part to a lowering of pH by formation of lactic acid since acidification of the blood with lactic, citric or hydrochloric acid also caused substantial reduction in autohemolysis.

Glucose caused slight to marked increase in lysis of red cells in certain othercases of spherocytosis, notably in autoimmune hemolytic disease and in myeloidmetaplasia. Addition of lactic acid to the blood from several of these patientshad similar effect.

In 2 atypical cases of chronic spherocytosis, with hematologically normalrelatives, autohemolysis was accelerated by addition of glucose to the bloodsamples. Adenosine had little effect until after splenectomy when both glucoseand adenosine inhibited hemolysis, much the same as in blood from typicalcases of hereditary spherocytosis.

It seems likely that when the abnormalities of carbohydrate metabolism ofthe red cells in certain hemolytic disorders are better understood, measurementsof autohemolysis may be modified so as to enhance their usefulness in detectingand differentiating the various hemolytic states. It also seems likely that furtherstudies on in vitro autohemolysis will help to elucidate some hemolytic mechanisms operating in vivo.

Submitted on March 14, 1956 Accepted on April 17, 1956     

Hereditary Spherocytosis: Observations on Hemolytic Mechanisms and Iron Metabolism

1. Two healthy patients with hereditary spherocytosis were phlebotomizeduntil they developed iron deficiency and the erythrocytes became hypochromic.The hereditary spherocytes were no longer spheroidal: they became thin, andthe fragility tests improved. However, the life span of the cells in the circulation was not improved. Later, splenectomy corrected the hemolytic disease.

2. In both patients, prior to the experiment, the hemolytic disease wascompensated. There was no anemia despite the rapid turnover of red cells.In one of the patients, whose average red cell life span was only five days, theoutput of hemoglobin must have been exceedingly high. It was computed tobe 135 Gm. per day, or 20 times the normal rate.

3. Some aspects of iron metabolism in hereditary spherocytosis are discussed.

4. The shape of the red cell in HS does not appear to be responsible for itspremature destruction by the spleen. Iron deficiency corrects the spherocytosis,but it does not correct the hemolytic disease. Splenectomy corrects the hemolytic disease, but it does not correct the spherocytosis.

     

Fast Hemoglobin in Lead Poisoning

1. An electrophoretically fast hemoglobin was found in approximately 40per cent of preschool children with elevated blood lead levels.

2. Fast hemoglobin was found more often in lead-poisoned patients withhypochromic anemia than in patients with normochromic red cells.

3. Fast hemoglobin differed from hemoglobins produced in vitro by incubation with chromate or oxidized glutathione. It had electrophoretic propertiessimilar to that found in a few patients receiving tolbutamide.

4. Fast hemoglobin could not be differentiated from normal hemoglobin A₃by any technic utilized.

5. Both lead and A₃ hemoglobins were heterogeneous molecular species.

6. The mechanisms leading to the production of hemoglobin A₃ and leadhemoglobin remain unknown.

Submitted on October 7, 1965 Accepted on December 10, 1965     

The Hemolytic Anemia of Human Bartonellosis

A study of the processes of formation and destruction of blood has beencarried out, in addition to other investigations of the physiopathology of theanemia of human bartonellosis. From the results obtained the following conclusions may be drawn:

1. The life-span of the red cells parasitized by Bartonella bacilliformis isgreatly shortened. However, not all the parasitized red cells are prematurelydestroyed.

2. Red cells from normal subjects are partially destroyed when they are injected into infected patients. More than 50 per cent of them survive normally.

3. The mechanical fragility of the red cells is increased in the majority ofthe cases.

4. The index of sequestration of red cells by the liver and spleen was increased in the three patients studied. Also, the products of catabolism of hemoglobin were increased in all the patients studied.

5. The increased production of red cells as a response to the great destructionwas prevented at first, but later it reached its peak, being in some cases fivetimes greater than normal.

6. The search for agglutinins and hemolysins was negative.

7. The amount of free protoporphyrins in the red cells was increased, indicating that there was some interference to the synthesis of hemoglobin thatwould also explain the hypochromia of the red cells.

8. The increase in the diameter of the red cells was independent of the actualamount of reticulocytes.

Submitted on November 23, 1960 Accepted on February 12, 1961     

The effects of splenectomy on the red blood cells of the dog with particular emphasis on the reticulocyte response

In splenectomized dogs the following observations on the erythroid elementswere made:

1. There was no unusual postoperative depression of erythrocyte, hemoglobin or hematocrit values, and regeneration occurred at the same rate as inthe control dogs.

2. A significant increase in the number of circulating reticulocytes wasnoted, which persisted for a longer duration than the associated thrombocytosis. There is a suggestion that the reticulocytes circulating during regeneration were more immature than those of the control animals.

3. Varying numbers of target cells accompanied by increased osmotic resistance were present in all the splenectomized dogs. Most also developednormoblastemia, particularly during active regeneration, which persistedthereafter in some of the animals. Howell-Jolly bodies were rare.

4. No significant bone marrow changes were noted.

It is suggested that:

1. In nonsplenectomized animals, most erythrocytes released from the bonemarrow in response to blood loss are mature red cells rather than reticulocytes.

2. Postsplenectomy reticulocytosis is not dependent on a fall in hematocritand is not a reflection of hemolysis or even of increased erythropoiesis, except,perhaps, in part during regeneration from the blood loss incurred at surgery.

3. Postsplenectomy reticulocytosis and normoblastemia are unaccompaniedby alterations in the myeloid-erythroid ratio of the bone marrow and areprobably largely due to a diminution of the normal inhibition of erythroid release from the marrow, permitting their earlier entry into the circulation.

4. The aforementioned changes are due specifically to the removal of thespleen itself, rather than merely to the removal of splenic blood from theportal circulation.

Submitted on November 18, 1957 Accepted on April 12, 1958     

The Relationship of Tumor Necrosis to Red Blood Cell Changes in the Hamster

1. Sterile, cell-free saline extracts of the necrotic portion of methylcholanthrene-induced hamster sarcoma injected into non-tumor bearing hamstersproduced a statistically significant decrease in hemoglobin, hematocrit andred blood cell levels.

2. These changes in hemoglobin, hematocrit and red blood cells arecomparable to those observed during sarcoma growth.

3. Sterile, cell-free, extracts of viable sarcoma injected into hamsters failedto produce alterations in the hemoglobin, hematocrit or red blood cell values.

4. The necrosis produced during sarcoma growth in the hamster cheekpouch is concluded to be responsible for alterations in the red blood cellcounts, hemoglobin and hematocrit values.

Submitted on December 10, 1958 Accepted on January 27, 1959